Compositions and methods for inhibiting photoaging of skin

ABSTRACT

Compositions and methods are provided for ameliorating various effects of UVA and UVB radiation from the sun. The compositions including an ingredient that prevents photoaging from MED and subMED radiation, such as a retinoid, certain other compounds (such as N-acetylcysteine, 2-furildioxime, and vitamin C) and optionally other MMP inhibitors such as tetracyclines and/or compounds that inhibit the P-450-mediated metabolism of retinoids such as ketoconazole and other azole compounds. In the method, the composition is applied prior to exposure to the sun; depending upon the ingredients used in the composition, application should be from 7 to 48 hours prior to exposure. Compounds that prevent erythema (skin reddening, sunburn) do not necessarily protect against UV-mediated elevation of MMP levels and activity, and similarly compounds that prevent UV-mediated elevation of MMP levels and activity are not necessarily effective against UV-induced erythema.

This is a divisional application of Ser. No. 09/089,914 filed Jun. 3,1998 which claims benefit of Ser. No. 60/048,520 filed Jun. 4, 1997 andclaims benefit of Ser. No. 60/057,976 filed Sep. 5, 1997.

TECHNICAL FIELD

This invention involves photoprotection of human skin. Moreparticularly, the invention relates to compositions for topicalapplication and to methods for using the same to inhibit photoaging ofskin, especially as induced by exposure to incidential and/or directradiation as would occur daily. Separately, this invention providesnovel methods and compositions for reducing UV-induced erythema (skinreddening).

BACKGROUND

Photoaging is a term presently used to describe the changes inappearance and/or function of human skin as a result of repeatedexposure to sunlight, and especially regarding wrinkles and otherchanges in the appearance of the skin.

Solar radiation reaching the earth's surface that effects and enablesvarious animals, including humans, comprises ultraviolet (UV) (λ<400nm), visible (400 nm<λ<700 nm), and infrared (IR) (λ>700 nm). UVradiation is generally divided into UVA (320-400 nm), UVB (290-320 nm),and UVC (<290 nm); UVC radiation is blocked from reaching the earth'ssurface by stratospheric ozone. The ultraviolet (UV) component ofsunlight, particularly UVB, is generally believed to be the principalcausative agent in photoaging.

The extent of UV exposure required to cause photoaging is not currentlyknown, although the amount required to cause erythema (reddening,commonly seen as sunburn) in human skin is known and quantifiedempirically as the “minimal erythemal dose” (“MED”) from a given UVsource. UVB wavelengths of 290-300 nm are the most erythmogenic. Theeffectiveness of UV radiation in causing erythema decreases rapidly asthe UV wavelength is increased beyond about 300 nm; wavelengths of 320nm and 340 nm are, respectively, one hundred and one thousand times lesspotent at causing skin reddening than wavelengths of about 298 nm.Repeated exposure to sunlight at levels that cause erythema and tanningare, nevertheless, commonly associated with photoaging. Erythema fromUVB is suggested to be a function of the total radiation exposure, notthe intensity of the radiation exposure. According to Physiology,Biochemistry, and Molecular Biology of the Skin, 2nd Ed., ed. by L. A.Goldsmith (New York: Oxford Univ. Press, 1991), UVA is considered bothmelanogenic and erythemogenic and UVA exposure induces synthesis of a 32kDa stress protein in human skin, as well as immediate erythema notapparent after UVB exposure.

Photoaging in human skin is characterized clinically by coarseness,wrinkles, mottled pigmentation, sallowness, laxity, eventuallypremalignant, and ultimately malignant neoplasms. Photoaging commonlyoccurs in skin that is habitually exposed to sunlight, such as the face,ears, bald areas of the scalp, neck, forearms, and hands.

Sunscreens are commonly used to prevent photoaging of skin areas thatare exposed to sunlight. Sunscreens are topical preparations thatcontain ingredients that absorb, reflect, and/or scatter UV light. Somesunscreens are based on opaque particulate materials, among them zincoxide, titanium oxide, clays, and ferric chloride. Because suchpreparations are visible and occlusive, many people consider theseopaque formulations cosmetically unacceptable. Other sunscreens containchemicals such a p-aminobenzoic acid (PABA), oxybenzone, dioxybenzone,ethylhexyl-methoxy cinnamate, octocrylene, octyl methoxycinnamate, andbutylmethoxydibenzoylmethane that are transparent or translucent on theskin. While these types sunscreens may be more acceptable cosmetically,they are still relatively short-lived and susceptible to being removedby washing or perspiration.

As noted above, the generally accepted etiology of photodamage to skininvolves an exposure to sunlight sufficient to cause erythema (sunburnor reddening; literally a flush upon the skin), and it is now known thatsufficient UVB radiation does cause erythema. This philosophy dictatesthat present compositions and methods for inhibiting photoaging includethe use compounds that block or absorb UVB, and that such compositionsneed be used only when there is sufficient likelihood that exposure tosunlight will result in erythema. More recent sunscreen compositionsinclude combinations of compounds that block both UVA and UVB radiation.

It has been suggested that UV solar radiation induces reactive oxygenspecies (ROS) in the skin. Rieger, M. M. Cosmetics and Toiletries (1993)108:43-56 reviews the topical application of known antioxidants to theskin for reducing the presence of ROS.

Retinoids have been used as therapy to improve the appearance ofsun-damaged skin. U.S. Pat. No. 4,877,805 describes the treatment ofphotoaged skin. The patent indicates that there is little point inbeginning the application of a retinoid to treat photodamage until theeffects of aging begin to appear. Several studies have investigatedimproving the appearance of existing photodamaged skin with the use ofall-trans retinoic acid. G. D. Weinstein et al., “Topical Trentinoin forTreatment of Photodamaged Skin,” Arch. Dermatol., 127:659-665 (May1991); J. S. Weiss et al., “Topical Tretinoin Improves Photoaged Skin,”J. Amer. Med. Assn., 259(4):527-532 (Jan 22/29, 1988).

Matrix metalloproteinases (MMPs) are a family of enzymes that play amajor role in physiological and pathological destruction of connectivetissue, especially collagen. Various types of collagen and collagenases(types of MMPs) are known in this field, and a further description canbe found in our copending U.S. patent application. Ser. No. 08/588,771,filed Jan. 19, 1996, the disclosure of which is incorporated herein byreference in its entirety and for all purposes. Inhibitors of MMPs(i.e., direct inhibitors of the proteinase) and of molecular pathways(i.e., inhibitors of AP-1) that affect MMP expression are known in otherfields and likewise are described in the aforementioned application No.588,771.

In summary, the state of the art considers that photodamage is causedprimarily by UVB radiation, and that presently available sunscreens aresufficient to prevent photodamage. “Dr. Ceilley [current President fothe American Academy of Dermatology] believes that staying out of thesun and using sunscreen could have prevented many of the skin cancersthat he treats in his practice, as well as the premature wrinkles thathis patients are concerned about.” Skin SAVVY, Amer. Acad. Dermat. supp.to USA Today, May 1997.

SUMMARY OF THE INVENTION

The present invention is based, in one preferred embodiment, on ourdiscovery that suberythemal doses of UV radiation induce MMPs thatdegrade skin connective tissue and thus are likely responsible forphotoaging. That is, we have discovered that UV radiation exposuresinsufficient to cause erythema nevertheless induce MMPs which degradedermal connective tissues, such as collagen and elastin, presumed tocause photodamage. That is, a UV exposure (with UVA and/or UVB)insufficient to cause erythema nevertheless is sufficient to causephotodamage via MMP induction. As such, the term “photodamage” should beredefined in the art so as not to require erythema. Thus, a combinationof UVA and/or UVB radiation can significantly damage the skin. Ourinvention broadly includes preventing photodamage from UVA and/or UVBradiation, especially before clinical signs of photodamage arepresented.

In our preferred embodiments, retinoids are used to prevent photodamage.In another embodiment of this invention, we have found that variousother compounds are useful in preventing photodamage by inhibiting theproduction and/or activity of MMPs. Though some of these compounds aretermed “antioxidants” and may prevent erythema, they also may reduce theconcentration of MMPs in UV-exposed human skin. Separately, our resultstesting such compounds show that prevention of erythema does notcorrelate with inhibiting UV-mediated increases in MMPs.

In yet another embodiment of this invention, we have found thatretinoids can inhibit the elevated MMP levels due to UV-exposure onhuman skin. While the prior art teaches that retinoids are useful forthe treatment and repair of photodamaged skin, we have discovered thatretinoids can interfere with the UV-induced elevation of MMP levels, andso retinoids can be used prophylatically to prevent photodamage fromoccuring.

In summary, then, one embodiment of our invention comprises acomposition, especially for daily use, comprising a UVA blocker, a UVBblocker, and an MMP inhibitor in a topically acceptable carrier.

Also included is a method for preventing incidental photodamage, aprophylactic against photodamage where incidental UV exposure does notproduce erythema, by the topical application of a composition comprisinga UVA blocker, a UVB blocker, and an MMP inhibitor, to normally exposedskin (such as the face, head, hands, and forearms).

Yet another embodiment of our invention is a composition, especially forprophylactic use against photodamage, comprising an erythema inhibitorand an MMP inhibitor.

In another embodiment our invention is directed to window structureshaving a coating thereon or admixed therewith a UVA blocker and a UVBblocker. As used herein, a blocker is broadly a compound that blocks thedirect effects of radiation on the skin by absorbing, reflecting, ormodulating to a non-harmful wavelength the particular light.

In still another embodiment of this invention, in those compositions inwhich a retinoid is present, the composition preferably furthercomprises a compound that inhibits the breakdown of the retinoid in theskin. Such compounds are those that inhibit the cytochromeP-450-mediated breakdown of retinoids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is depicts what is believed to be the general pathways for skindamage due to UV exposure based on our discoveries.

FIG. 2 depicts evidence that suberythemal UVB/UVA radiation inducescollagenase, stromelysin-1, and the 92 kDa gelatinase, all MMPs; thehistogram is a quantitive representation of the radioblot test for eachof these proteins.

FIG. 3 depicts evidence that regularly repeated suberythemal UVB/UVAexposure of human skin induces consistently elevated levels of the 92kDa gelatinase and collagenase MMPs.

FIG. 4 depicts the spectrum emitted by an illumination apparatus, bothunfiltered and through five different filters, used for the resultsdepicted in FIG. 5.

FIG. 5 depicts the induction of collagenase in human skin as a functionof UV wavelength (through the various filters shown in FIG. 4) wherein aconstant amount of energy was delivered.

FIG. 6 depicts the spectrum emitted by the illumination apparatus usedfor the experiments herein (except that having results shown in FIG. 5).

FIG. 7 depicts the results topically applied commercial sunscreens hadon erythema induction after 2 MED UVB/UVA exposure of human skin.

FIGS. 8A and 8B depict the UV absorbance of a UVA blocker and the effectof pretreatment with such blocker on erythema in UV-exposed human skin.

FIG. 9 depicts the effects of melatonin, vitamin E, N-acetyl cysteine(NAC), and 2-furildioxime (FDO) on preventing erythema from exposure totwo MEDs of radiation, and of acetylsalicylic acid (ASA) and vitamin Con preventing erythema from exposure to one MED of radiation in humanskin.

FIG. 10 depicts the effects of melatonin, vitamin E, N-acetyl cysteine(NAC), and 2-furildioxime (FDO) on preventing elevated collagenaseactivity from exposure to two MEDs of radiation, and of acetylsalicylicacid (ASA) and vitamin C on preventing elevated collagenase activityfrom exposure to one MED of radiation in human skin.

FIGS. 11A-11B depict the effect of the time of pretreatment of skin withNAC on the efficacy of NAC to inhibit UV-induced collagenase andgelatinase.

FIGS. 12 depicts the effectiveness of a particular UVA blocker forinhibiting UV-induced 92 kDa gelatinase in human skin.

FIG. 13A depicts the effect of pretreatment with retinoids on theinhibition of UV-induced elevations in collagenase, the 92 kDagelatinase, and stromelysin-1 in human skin upon exposure to 2 MEDs ofradiation.

FIG. 13B depicts the effect of two different retinoids on subMEDUV-induced increase in type I collagenase activity.

FIGS. 14A-14D depict the time course of the elevation of various MMPs(respectively collagenase, stromelysin, 92 kDa gelatinase, and 72 kDagelatinase) in human skin after exposure to UV radiation.

FIGS. 15A-15B depict the effect of the time of pretreatment on theeffectiveness of retinoids for inhibiting UV-induced collagenase and the92 kDa gelatinase, and of c-JUN protein.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention is directed to inhibiting (i.e., reducing orpreventing) photoaging of skin, especially human skin. Treatmentaccording to this invention is preferably practiced on skin such as thatof the head, neck, hands, and arms that in typical, everyday living arehabitually exposed to sunlight. Because repeated exposure to doses of UVbelow those that cause erythema can lead to photoaging, the inventionshould be practiced on skin subject to such low dose exposure.

UVB doses in the range of 30-50 mJ/cm² skin cause erythema in mostfair-skinned people. Accordingly, the invention will prevent photoagingof skin subjected to doses below this range (typically above about 5mJ/cm² which is equivalent to a few minutes of sunlight exposure).Sunlight reaching the surface of the earth when the sun is essentiallyoverhead provides the following amounts of radiation: 0.5% UVB; 6.5%UVA; 38.9% visible light; and 54.0% IR. These radiation types providethe following energy fluxes: 2.11 mJ/cm²·s (21.1 W/m²) for UVB; 8.57mJ/cm²·s (85.7 W/m²) for UVA; 53.2 mJ/cm²·s (532 W/m²) for visiblelight; and 72.2 mJ/cm²·s (722 W/m²) for IR.

While not desirous of being constrained to a particular theory, thefollowing examples may be better appreciated with reference to FIG. 1,which depicts an overview of some of the UV-induced biochemical pathwaysleading to changes in the skin. As shown, UV radiation induces a MAPkinase cascade from which two resulting pathways are shown: one resultsin induction of interlukins, which lead to erythema; the other resultsin induction of MMPs, which lead to connective tissue degradation. Whilethe art has considered these results as due to UVB radiation, we havenow shown in a series of experiments that UVA is a definitive culprit inphotodamage even when UV-exposed skin provides no visual clues ofphotodamage. It should be understood in connection with thisapplication, however, that both UVA and UVB are responsible, possiblyeven independently, for UV-mediated induction of MMPs in human skinafter exposure to solar radiation.

Exposure to UV radiation is typically measured in these arts byreference to the minimal erythemal dose, MED, which is defined as anexposure to UV radiation sufficient to cause reddening of the skin. One(1) MED is equivalent generally to about 30 mJ/cm²·s of solar radiation.The philosophy of the prior art is that exposure to natural sunlightsufficient to cause redness (sunburn, erythema) initiates photoaging.Using the UV source described below, which emits both UVA and UVBradiation (with a lower ratio of UVA/UVB than found in naturalsunlight), we have confirmed that if skin redness is induced then MMPsare also induced. Thus, the present philosophy of the art is thatsunscreens should be used because they prevent redness and so preventphotoaging.

We believe we have contradicted some of the present philosophy and havealso found unexpectedly the effect on human skin of various UV exposuresand the use of various compounds applied prior to exposure. One of ourunexpected results is that UV exposure insufficient to cause skinredness nevertheless induces increased MMP activity (and so photoaging)in human skin. Thus, conventional sunscreens may prevent redness but maynot prevent photoaging from the increase in MMP activity after UVexposure. We have also identified compounds that prevent redness, whichis important not only to prevent the pain and discomfort caused byerythema, but also possibly for compliance by including an erythemainhibitor in a composition that inhibits photoaging (because a patientmay tend to believe the antiphotoaging component of the composition isnot effective if erythema results after use of a composition touted aspreventing photodamage).

Another unexpected finding is that blocking UVA radiationprophylactically inhibits both increased MMP activity and increased cJUNprotein concentration in UV-exposed human skin, and so is a prophylacticagainst photoaging. Combined with our finding that suberythemal UVexposure causes photoaging, one aspect of our invention contemplates thedaily use of a UVA blocker as a prophylactic against photoaging. BecauseUVB also induces MMPs, a more preferred composition would include both aUVA and a UVB blocker.

Still another unexpected finding is that pretreatment of skin with aretinoid mitigates the increased MMP activity typically occurring afterUV exposure. Accordingly, our invention contemplates a composition fortopical application prior to UV exposure that contains a retinoid as aprophylatic against photoaging.

Yet another unexpected finding of our investigations is that certaincompounds (some having been reported to have antioxidant properties)provide a good anti-erythemal sunscreen effect, although they do notappear to inhibit increased MMP activity subsequent to UV exposure.

The invention is now described with reference to the figures. Thedetails of the experiments from which the results shown in the figureswere obtained, as well as the apparatus used to irradiate our humanvolunteers, and the immunohistological methods are described below. Thearea of the volunteers' skin tested is typically hidden or physicallyprotected from the sun exposure during most of one's life (e.g., skinfrom the hips and buttocks). As noted from the results of ourinvestigation, one cannot rely solely on in vitro experimentation todetermine whether a compound is an MMP inhibitor and is also suitablefor use as an inhibitor in vivo. Accordingly, one must test suchcompounds by methods as described herein to determine whether suchcompounds provide the desired therapeutic effect.

FIG. 2 depicts evidence that suberythemal UV exposure induces thecollagenase, stromelysin-1, and the 9 skDa gelatinase MMPs. Portions ofvolunteers' skin was exposed to the following amounts of UV radiationexpressed as a fraction of one (1) MED: 0.01, 0.05, 0.1, 0.5, 1, and 2.Biopsies and subsequent radioassays reveal (as shown in the radioblot inthe figure, which is represented quantitatively by the histogram) thatone-half of an MED is sufficient to induce MMPs; even 0.1 MED issufficient to elevate the production of MMPs significantly abovebaseline levels; and 0.01 MED is sufficient to elevate collagenase abovethe baseline level. Thus, FIG. 2 shows that suberythemal UV radiationcauses the production of MMPs. Nevertheless, it might be assumed thathuman skin returns to a baseline state where the levels of MMPs are notelevated, especially after exposure to low doses of UV radiation.

FIG. 3 presents further evidence that repeated exposure to suberythemalUV radiation generates MMPs and that these levels remain elevated overtime. When people were irradiated with one-half MED every two days, thelevel of MMPs remained elevated, and so collagen is continuously brokendown by repeated, subMED exposure to UV radiation. FIG. 3, combined withthe knowledge that very small UV doses induce MMPs as shown in FIG. 2,implies that daily, subMED, yet chronic exposure to UV radiation causeselevated MMP levels in human skin, and thus one's skin may never fullyrecouperate from chronic subMED UV exposure.

FIG. 4 depicts the spectrum emitted from the illumination apparatusunfiltered and with various conventional filters (WG320 1; F1 3 mm; UV342.5; SF12 2; and WG360 2.5). The spectrum emitted from the apparatusthrough the various filters is shown by the different types of lines.The WG320 1 filter can be considered to approximate the sun with bothUVB, UVA2, and UVA1 radiation, whereas the WG360 2.5 filter allows onlyUVA1 radiation to pass through. This apparatus includes both UVB lamps(Philips model TL40W/12/RS, available from Ultraviolet Resources Inc.,Lakewood, Ohio) and UVA lamps (Q-Panel UVA-351, available from Q-PanelLab Products, Cleveland, Ohio).

We tested for the induction of collagenase as a function of wavelengthwhen the person was irradiated with a constant amount of energy. Giventhe curves shown in FIG. 4, the relative durations during which avolunteer was exposed under a given filter can be determined by theratio of an integration of the areas under each of the curves as afunction of the wavelengths emitted; thus, even though the same amountof energy was delivered to the subject, the duration of the exposureunder the WG360 2.5 filter was longer than under the WG320 1 filter. Theresults shown in FIG. 5 imply that a combination of UVB and UVA inducescollagenase (filter WG320 1), and that UVA1 alone (filter WG360 2.5) isalso sufficient to induce collagenase. At early and late times of daywhen the sun is low on the horizon, the proportion of UVA to UVB isactually increased, and so skin is exposed to more UVA radiation than itwould be at noon time. Thus, the results shown in FIG. 5 that UVA1 issufficient to cause elevated MMPs, which occurs at early and late timesof day when sun exposure does not cause erythema, indicates thatphotodamage still occurs at those times of day, even in the absence oferythema. Also, contrary to what the average person would consider to bea “safe” time of day to be out in the sun because sunburn is unlikely tooccur, nonetheless is not safe because MMPs still can be induced by thesun's UV radiation.

The erythema response is of clinical importance because, at the veryleast, significant pain and discomfort occurs. Various over-the-countersunscreens do provide protection against erythema, as shown in FIG. 7.Typically these sunscreen contained only a UVB blocker, although manyare now marketed with a “UVA” blocker. The blocking spectrum of acommercially available UVA blocker, PARSOL 1789, is shown in FIG. 8A;the right hand vertical scale correlates with the absorbancecharacteristics of the blocker, and left vertical scale correlates withthe relative effectiveness of the blocker in preventing erythema atwavelengths generally greater than about 300 nm. This UVA blocker doesprovide some protection against 2 MED from our standard source (FIG.8B).

Various other compounds have been used to prevent erythema. We tested anumber of different compounds, which were applied to skin about sevenhours prior to UV exposure and subsequent biopsy, for the effectivenessin preventing UV-induced skin redness. Melatonin does appear to preventerythema at irradiation doses of above about 2 MED. Vitamin E wasslightly worse but still very effective at preventing erythema.Acetylsalicylic acid (ASA) and vitamin C also provided protectionagainst erythema induced from one MED when applied 16 hours prior toexposure. FDO is also effective at preventing erythema. NAC apparentlyhad no effect against erythema.

Based on the results shown in FIG. 9, one of our inventions is a methodfor preventing erythema by applying to skin that will be exposed to UVradiation (i) melatonin and/or vitamin E (or a derivative of either) atleast about 7 hours prior to exposure, and/or (ii) acetylsalicylic acid,vitamin C, and/or FDO (or a derivative of any thereof) at least 16 hoursprior to exposure.

Having investigated and described various anti-erythematic compounds(FIG. 9), these compounds were tested to determine if prevention oferythema was indicative of prevention against elevated levels of MMPs,the results of which are shown in FIG. 10. Although good at preventingsunburn, neither melatonin nor vitamin E (both with pretreatment)prevented induction of MMPs such as the 92 kDa gelatinase andcollagenase after exposure to 2 MEDs. Likewise, although useful atpreventing sunburn, ASA was not effective at preventing elevatedcollagenase activity. Unexpectedly, NAC, which was not effective atpreventing erythema, was effective at preventing increased MMP activityafter exposure to two MED. We also discovered that the use of NAC toprevent increased MMP levels (such as the 92 kDa gelatinase andcollagenase) requires pretreatment for more than four hours, andpreferably at least about seven hours prior to exposure (one MED; FIGS.11A and 11B).

In retrospect, compounds that prevented an erythemogenic response(melatonin, vitamins C and E, FDO, and ASA) were not necessarily alsoeffective at preventing a UV-induced increase in collagenase activity(comparing FIGS. 9 and 10). On the other hand, compounds apparently noteffective for preventing erythema (e.g., NAC) can be useful forpreventing the UV-mediated increase in MMPs. Thus, another invention isthe use of NAC, FDO, or vitamin C (or a derivative of any) to preventUV-induced elevation of MMPs such as the 92 kDa gelatinase andcollaganese, applied at least about seven hours prior to exposure.

Having shown above that UVA1 induces elevated levels of MMPs, UVA1blockers are also useful at preventing this elevation (FIG. 12). Theseblockers may prevent initiation of the pathway(s) leading to increasedMMP levels and/or activity, as they also prevent induction of c-JUNprotein (data not shown).

Retinoids are preferred inhibitors of UV-induced increases in the levelsand/or activity of MMPs. Retinoic acid decreases 2 MED UVB-mediatedinduction of the levels and activity of the MMPs collagenase, 92 kDagelatinase, and stromelysin-1, as well as their transcription (measuredas mRNA) when applied 48 hours prior to exposure (FIG. 13A).Approximately ten times the concentration of retinol is about aseffective as retinoic acid at preventing UV-induced elevation in type Icollagenase activity, even at suberythemal radiation doses (FIG. 13B).

A single two MED UV exposure leads to increased MMP levels which aretypically maximal about 24 hours after exposure (FIGS. 14A-14A; the sameas FIGS. 2a-2 d in our copending application 588,771). As with thevarious compounds found effective against erythema or UV-induced MMPactivity, pretreatment is preferred when using retinoids, and theearlier the pretreatment before exposure, the better (FIG. 15A). Longertreatment times prevent, over time, the UV-mediated increase in c-JUNprotein levels, which presumably lead to the increased MMP levels. Infact, the elevation in c-JUN protein levels appears to be severelylimited when a retinoid is used about 48 hours prior to exposure (FIG.15B). Although one might expect the time course of the levels of c-JUNprotein to mirror the time course of those of the MMPs induced by UVexposure, those levels remain at a constant and only slightly elevated(compared with baseline, although they are signicantly below the levelsinduced in untreated, unprotected skin) when a retinoid is used as anMMP inhibitor. The decreased levels of c-JUN protein indicates that theretinoid decreases the production of MMPs over the entire time coursestudied rather than changing the kinetics of the UV-mediated skinreaction.

The present invention includes as a method for preventing photoaging ofskin the daily topical application of a composition having both an MMPinhibitor and UVA/B blockers. As shown herein, and contrary to thepresent philosophy of this medical art, suberythemal UV exposure causesthe generation of destructive proteinases. The vast majority of peopledaily spend some time in the daylight (be it walking the dog or walkingto work), and because this is not the conventional “sun bathing”, itwould not have been expected that daily, suberythemal exposure to thesun causes photodamage as the result, in part, of UV-mediated increasesin MMP activity. While a paleobiological explanation might be offeredwhy human skin functions to create MMPs upon suberythemal UV exposure,our method of preventing, or at least inhibiting, at least this type ofphotoaging can be accomplished by the daily topical application of (i) aUVA/B blocker (i.e., broadly one or more compounds that block the directeffects of UVA/UVB radiation on the skin by absorbing, reflecting, ormodulating the light to a non-harmful wavelength), (ii) a compoundprophylactically effective to inhibit or reduce UV-induced MMP activityincrease and/or a direct inhibitor of MMPs, and (iii) a compatiblemixture of one or more of these ingredients. In view of theseexperiments, a preferred embodiment of our invention is an improvedsunscreen composition which further comprises an MMP inhibitor,preferrably a retinoid, and both a UVA blocker and a UVB blocker.

As used herein, “inhibitors” of MMPs inhibit one or more of the steps inthe natural physiological pathways leading to MMP production and/ordirectly inhibit one or more of these proteinases. Thus, an MMPinhibitor can inhibit one or more of the various signalling compoundsand/or of the transcription factors (e.g., cJUN and cFOS, which togetherlead to the production of MMPS) by which MMPs are produced naturally.

Retinoids are one class of MMP inhibitors. The inhibitors of MMPs canact directly on the MMPs and/or on the transcription factors AP-1 andNF-κB by which MMPs are produced naturally. E55 10 has been described(by Fujimori, T., et at., Jpn. J. Pharmacol. (1991) 55(1):81-91) asinhibiting NF-κB activation. Retinoids such as those disclosed in U.S.Pat. No. 4,877,805 and the dissociating retinoids that are specific forAP-1 antagonism (such as those described by Fanjul, et al. in Nature(1994) 372:104-110), glucocorticoids, and Vitamin D₃ target AP-1.Compounds for enhancing the therapeutic effect of Vitamin D₃ may alsoenhance the MMP-inhibitory effect of Vitamin D₃ and such are describedin copending application Ser. No. 08/832,865 (J. Voorhees et al.,“Method for Assessing 1,25(OH)₂D₃ Activity in Skin and for Enhancing theTherapeutic Use of 1,25(OH)₂D₃”), filed Apr. 4, 1997, the disclosure ofwhich is incorporated herein by reference. Other retinoids, besidesretinol, include natural and synthetic analogs of vitamin A (retinol),vitamin A aldehyde (retinal), vitamin A acid (retinoic acid (RA)),including all-trans, 9-cis, and 13-cis retinoic acid), etretinate, andothers as described in EP-A2-0 379367, U.S. Pat. No. 4,887,805, and U.S.Pat. No. 4,888,342 (the disclosures of which are all incorporated hereinby reference). Various synthetic retinoids and compounds having retinoidactivity are expected to be usefull in this invention, to the extentthat they exhibit retinoid activity in vivo, and such are described invarious patents assigned on their face to Allergan Inc., such as in thefollowing U.S. Pat. Nos. 5,514,825; 5,698,700; 5,696,162; 5,688,957;5,677,451; 5,677,323; 5,677,320; 5,675,033; 5,675,024; 5,672,710;5,688,175; 5,663,367; 5,663,357; 5,663,347; 5,648,514; 5,648,503;5,618,943; 5,618,931; 5,618,836; 5,605,915; 5,602,130. Still othercompounds described as having retinoid activity are described in otherU.S. Pat., Nos.: 5,648,563; 5,648,385; 5,618,839; 5,559,248; 5,616,712;5,616,597; 5,602,135; 5,599,819; 5,556,996; 5,534,516; 5,516,904;5,498,755; 5,470,999; 5,468,879; 5,455,265; 5,451,605; 5,343,173;5,426,118; 5,414,007; 5,407,937; 5,399,586; 5,399,561; 5,391,753; andthe like, the disclosures of all of the foregoing and following patentsand literature references hereby incorporated herein by reference.

MMPs are also inhibited by BB2284 (described by Gearing, A. J. H. etal., Nature (1994) 370:555-557), GI129471 (described by McGeehan G. M.,et al., Nature (1994) 370:558-561), and TIMPs (tissue inhibitors ofmetalloproteinases, which inhibit vertebrate collagenases and othermetalloproteases, including gelatinase and stromelysin). Still othercompounds useful for the present invention include hydroxamate andhydroxy-urea derivatives, such as Galardin, Batimastat, and Marimastat,and those disclosed in EP-A1-0 558635 and EP-A1-0 558648 (as useful forinhibiting MMPs in the treatment of, among other etiologies, skinulcers, skin cancer, and epidermolysis bullosa). Retinoids have beenreported by Goldsmith, L. A. (Physiology, Biochemistry, and MolecularBiology of the Skin, 2nd. Ed. (New York: Oxford Univ. Press, 1991),Chpt. 17) to cause an increase in steady state levels of TIMP mRNA thatwould suggest transcriptional control; although, based on ourdiscoveries, we have found this is not true in human skin in vivo.

Other MMP inhibitors include genistein and quercetin (as described inU.S. Pat. No. 5637703, U.S. Pat. No. 5665367, and FR-A-2,671,724, thedisclosures of which are incorporated herein by reference) and relatedcompounds, as well as other antioxidants such as NAC (N-acetyl cystein),and others.

In addition to retinoids as a class of compounds useful for thisinvention, any drug which inhibits the cytochrome P-450 enzymes thatmetabolize retinoic acid can also be useful in practicing thisinvention. In the skin, retinoids are converted into retinoic acide (RA)as the active form. Natural retinoids that function in the skin are alltrans or are metabolized to all trans. Retinoic acid (RA; all trans) ismetabolized to inactivation by hydroxylation (via RA 4-hydroxylase) to4-hydroxy-RA, which is then oxidized by a reaction mediated by thecytochrome P-450-dependent monooxygenase system. (S. Kang et al.,“Liarozole Inhibits Human Epidermal Retinoic Acid 4-Hydroxylase Activityand Differentially Augments Human Skin Responses to Retinoic Acid andRetinol In Vivo,” J. Invest. Dermatol., 107:183-187 (Aug. 1996); E. A.Duell et al., “HumanSkin Levels of Retinoic Acid and CytochromeP-450-derived 4-Hydroxyretinoic Acid after Topical Application ofRetinoic Acid In Vivo Compared to Concentrations Required to StimulateRetinoic Acid Receptor-mediated Transcription In Vitro,” J. Clin.Invest., Skin Retinoid Levels and Reporter Gene Activity, 90:1269-1274(Oct. 1992); E. A. Deull et al., “Retinoic Acid Isomers Applied to HumanSkin in Vivo Each Induce a 4-Hydroxylase That Inactivates Only TransRetinoic Acid,” J. Invest. Dermatol., 106:316-320 (Feb. 1996); thedisclosures of which are incorporated herein by reference). Accordingly,compounds which interfere with the elimination metabolism of all transRA, the active metabolite of topically applied retinoids such as 9-cisRA and 13-cis RA, will beneficially increase the amount of RA in theskin. Thus, preventing the degradation of natural (all trans) RA in theskin effectively increases its concentration, and so provides thebenefits described herein. Examples of compounds dermatologicallyacceptable and having or likely to have inhibitory effects on theP-450-mediated degradation of RA include azoles, especially triazoles,including, for example, ketoconazole (U.S. Pat. Nos. 4,144,346 and4,223,036), fluconazole (U.S. Pat. No. 4,404,216), itraconazole (U.S.Pat. No. 4,267,179), liarozole, irtemazole, and the like; compoundsrelated to these that may also be useful include, for example, diazinessuch as flucytosine. It would also be beneficial to use such cytochromeP-450 inhibitors in combination with a reduced amount of retinoid; theP-450 inhibitor decreases the metabolic elimination of the retinoid andso less retinoid is needed to achieve the same result. Still further,analytical methods are available for determining whether a givencompound inhibits the degradation of RA by applying the compound andtesting for changes in CRABP (cytoplasmic retinoic acid bindingprotein), which will have increased levels if the levels of RA are alsoincreased by the topical application of the test compound.

Still other inhibitors of MMPs that can be applied topically and areuseful in practicing the claimed invention include the tetracyclines andderivatives thereof, such as minocycline, roliteracycline,chlortetracycline, methacycline, oxytetracycline, doxycycline,demeclocycline, and the various salts thereof. Because of possibleallergic or sensitization reactions, the topical adminstration oftetracyclines should be monitored carefully for such untoward reactions.

Various compounds termed “antioxidants” are also useful as MMPinhibitors. While not desirous of being constrained to any particulartheory of operation, these compounds may quench or otherwise reduce freeradicals and reactive oxygen species which may initiate or lead to MMPinduction, such as via the MAP kinase cascade. These compounds includeglutathione and its precursors, such as N-acetyl cysteine (NAC) orglutathione ethyl ester, more broadly N-CH₃(CH₂)_(n)CO cysteine (whereinn is an integer from zero to eight, more preferably not more than 4),and related compounds and derivates thereof as described in U.S. Pat.No. 5,296,500 (the disclosure of which is incorporated herein byreference). These other MMP inhibitors include water-soluble compoundssuch as vitamin C and NAC, and FDO. Various other compounds that may actas MMP inhibitors include: lipid-soluble compounds such as β-caroteneand its derivatives or other carotenoids; glutathione and derivativesthereof (or of NAC); α-lipoic acid (1,2-dithiolane-3-pentanoic acid);selenium compounds such as Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one); isoflavones such as genistein (isoflavone), quercetin(flavon-3-ol), and pycnogenol (flavan-3-ol(s)); ergothioneine; saponin(e.g., from Polypodium leucotomos); ginkgo biloba extract(flavoneglycoside and terpenelactone) and feverfew (Chrysanthemumparthenium) extract (sesquiterpene lactone).

Various UV blockers are known in the paint and dye industry to preventpigment or color degradation of cars, homes, and clothing. Aparticularly preferred UVA_(½)-blocker for use on human skin is PARSOL®1789 (Schering-Plough), as well as those in the aforementioned U.S. Pat.No. 4,387,089 that describes the preparation of this UVA-blocker. Wehave found that true UVA blockers inhibit induction of cJUN mRNA and ofcollagenase and gelatinase.

The compositions of this invention can be provided in any cosmeticallysuitable form, preferably as a lotion or cream, but also in an ointmentor oil base, as well as a sprayable liquid form (e.g., a “hair” spraythat protects hair and scalp against UV damage, in a base that dries ina cosmetically acceptable way without the greasy appearance that alotion or ointment would have if applied to the hair). In addition, thecompositions contemplated by this invention can include one or morecompatible cosmetically acceptable adjuvants commonly used, such ascolorants, fragrances, emollients, humectants, and the like, as well asbotanicals such as aloe, chamolile, and the like. When used topically,retinoids are used preferably at concentrations of between about 0.05%and about 5%, more preferably between 0.1% and 1%. Retinoids and thevarious antioxidants described above can also be taken systemically,preferably by oral administration. When dosed orally, retinoids arepreferably administered in amounts from about 0.1 mg/kg (of body weight)to about 1 mg/kg or even more, all doses below that at which toxicity islikely; and antioxidants are preferably taken in “megadoses” (e.g., atleast 1 g/d of vitamin C, at least 1000 I.U. of one or moretocopherols).

In summary, our invention is broadly viewed as refocussing the conceptof preventing “photoaging” from preventing sunburn to preventing theincrease in MMP activity following UV exposure. Our invention providesprophylaxis against photoaging through one or more modes: blocking UVA/Bradiation at the level of the skin by use of a UVA/B blocking sunscreen;blocking the generation by UV radiation of reactive oxygen species inthe skin that initiate the MAP kinase cascade and MMP induction by thetopical application of an antioxidant; blocking the induction oftranscription factors leading to increased MMP activity after UVexposure by the topical application of a retinoid or an MMP inhibitor(as broadly defmed herein); directly inhibiting MMP activity by thetopical application of an inhibitor thereof; and/or by blocking thetransmission of UVA radiation through a window structure to human skinby providing in the structure, or in a coating on the structure, a UVAblocker.

In view of our discoveries, it is clear that UV radiation atsuberythemal doses causes skin damage. Thus, while prescription glassesand most sunglasses include UV-reflective or -absorbing materials orcoatings, another aspect of our invention is to provide UV-coatings,especially against transmission of UVA, on all types of glass, includingnot only prescription and sunglasses but also for windows for homes andoffices and automobiles. In addition, because jet airliners flyextremely high in the atmosphere, passengers situated near windows maybe exposed not only to UVA and UVB radiation, but also possibly to moredamaging UVC radiation. Given the present description of our invention,one or ordinary skill in the art related to window coatings couldreadily identify a UVA blocker and incorporate such into a film-formableor curable (e.g., paint-like) coating for joining or lamination to awindow structure. Thus, in another embodiment, this invention includestransparent and translucent polymeric structures having UV-reflectiveand/or -absorbtive coatings (especially UVA-blockers) therein and/orcompounds therein. Such structures include window-like andwindow-covering devices, such as plastic awnings for baby carriages andplastic shades (typically colored or tinted) hung up in store windowswhen the sun is low. Again, one of ordinary skill in the art offabricating these types of structures can now readily provide a UVAblocker, incorporate such into a film-forming polymeric material (e.g.,plasticized polyvinyl acrylate), and provide a transparent ortranslucent window structure that blocks the transmission of UVAradiation. In connection with UVA-blocking windows, as noted above therelative amounts of UVA and UVB change as a function of the sun'selevation in the sky. At lower elevations of the sun (i.e., the morningor evening sun, as opposed to the “midday” sun, zenith angle of 0°), therelative amount of UVA:UVB is increased compared with other times of day(e.g., noon). At these lower elevations, the relative amount of UVA toUVB can more than double. Thus, contrary to present suppositions thatthe midday sun causes the most damage, which suppositions are likelybecause the greater amount of UVB light at a higher zenith more easilycauses a bad sunburn, our discovery that a combination of suberythemalUVB plus UVA radiation causes photodamage shows the importance ofprotecting against photodamage at other times of day. Thus, a broadspectrum UVA/B window coating would be useful in protecting driversgoing to and/or from work each day in the morning and/or evening hours.

In the following examples, four F36T12 ERE-VHO UV bulbs were used toirradiate human skin. At all times, a Kodocel TA401/407 filter wasmounted 4 cm in front of the bulbs to remove UVC radiation (<290 nm).Radiation intensity was monitored using an IL443 phototherapy radiometerand an SED240/UVB/W photodetector (International Light, Newbury, Mass.).Spectroradiometry was performed using an Optronic Labroatories OL 754system. Total irradiance (290-800 nm) at about 43 cm (17 in.) from thesource of four bulbs was about 1.5 mJ/cm²·s (1.49×10⁻³ W/cm²). Theradiation output from this bulb was determined by spectroradiometry toprovide about 47% UVB and about 27% UVA (composed of about 9% UVA₁(340-400 nm) and about 18% UVA₂ (320-340 nm)), the remainder beingvisible and IR radiation. An exposure of about 160 seconds under thisset of four bulbs is equivalent to an exposure of one MED. Accordingly,when compared with natural sunlight which has 0.5% UVB and 6.5% UVA, itcan be seen that the set of four bulbs used in these experimentsprovides far less UVA radiation than would exposure to the sun of anequivalent amount of UVB.

In the following examples, a “standard vechicle” of 30% PEG(polyethylene glycol) in 70% ethanol (with 0.05% BHT as preservative)was used. UV-induced degradation of skin collagen was assessed byradioimmunoassay of soluble cross-linked telopeptides. mRNA and proteinlevels of MMPs and either endogenous inhibitors (TIMPs) were determinedby Northern and Western analyses, respectively. Collagenase activity wasmeasured by degradation of type I [³H] collagenase. MMP activities weremeasured by zymography.

EXAMPLE 1 Suberythemal Induction of AP-1

Nine caucasian adults were exposed on their buttocks region (i.e., skinnormally not exposed to sunlight) to the UV radiation from theaforedescribed set of bulbs for various times, after which tissuesamples were taken and analyzed. As shown in FIGS. 2 and 3, and usingthe aforementioned time of 2 minutes and 40 seconds (160 s) as one MED,various portions of these volunteers' skin were exposed to 0, 0.01,0.05, 0.1, 0.5, 1, and 2 MED of bulb radiation. The biopsied dermaltissue samples from exposed (and 0 MED, unexposed) skin were assayed forthe presence of AP-1 and the fold increase of binding to DNA encodingAP-1. As described by Angel, P., et al., Cell (1987) 49:729-739 andSato, H. and Seiki, M., Oncogene (1993) 8:395-405, the production ofcertain MMPs is mediated by the transcription factor AP-1.

The results of the biopsies shown in these figures are startling. Atsuberythemal doses down to about at least 0.01 MED, AP-1 is induced atlevels clearly greater than present in unexposed skin. These unexpectedresults lead us to believe that photodamage to human skin can be inducedby suberythemal MED radiation doses including UVB and UVA, andaccordingly humans everywhere can be protected against photoaging by thedaily application of a sunscreen that blocks at least UVA and optionallyalso UVB.

EXAMPLE 2 Retinoid Prophylaxis of Suberythemal Collagenase Induction

As described in our copending application Ser. No. 588,771 (referred toabove and incorporated herein by reference) it has been shown thatretinoids inhibit the induction of various MMPS, including collagenases,after erythemal doses of radiation.

Using the buttocks skin of ten volunteers, following the same generalprocedure as described above, each of these volunteers was pretreatedwith the standard vehicle alone, with 0.1% retinoic acid (RA), or with1% retinol (ROL). Tissue samples from these volunteers were biopsiedfollowing pretreatment and after no exposure and after exposure to 0.5MED from the set of four bulbs.

The results of these biopsies are shown in FIG. 13B, which depicts thepretreatment and exposure regime to the fold increase of type Icollagenase in vivo for the ten volunteers. As shown by the results inthis figure, pretreatment of human skin with a retinoid can inhibitsuberythemal UV-induced collagenase activity. Consistent with theresults shown in FIG. 2, suberythemal UV exposure causes a significantincrease in collagenase activity.

The results of Examples 1 and 2 were unexpected and intriguing to us,and prompted us to question the present philosophy of skin protectionand solar-induced skin damage. Throughout time, and in differentcultures, where the tanned, “outdoors” look is not consideredaesthetically appealing, such as in Elizabethan England and in manyOriental cultures (e.g., Japan, Korea), various compounds andcompositions have been tried to prevent sun damage to skin and/or toinduce a “protective” tan. We decided to test various compounds andcompositions, both old and new, for their true in vivo effect onUV-exposed skin.

EXAMPLE 3 Effect of UV Exposure after Topical Pretreatment I

Melatonin is a hormone apparently mediated by the light-dark cycle ofday-night. It has been proposed recently that melatonin might act as anantioxidant.

We evaluated six volunteers to determine the effect, if any, of topicalmelatonin on UV-induced erythema using the same general procedure asdescribed. The various UV dose exposures and the erythematic response ofeach of these volunteers after a five (5) minute exposure is depicted inFIG. 9. The previously unexposed skin of each of these volunteers waspretreated with the standard vehicle alone or with 5% melatonin. Theresults in FIG. 9 show that after exposure to two (2) MED, erythema wasinduced in vehicle-treated skin and was not induced in melatonin-treatedskin. Even when erythema was induced in melatonin-treated skin, it waspresent to a significantly lesser degree than in vechicle-treatedUV-irradiated skin. Nevertheless, it should be kept in mind, as shownabove, that lack of erythema does not necessarily correlate with lack ofphotodamage.

In another set of experiments, volunteers had areas of unexposed skinpretreated with vehicle alone or with 5% melatonin or with 5% vitamin Eabout seven hours prior to UV exposure. The areas were then exposed toabout 2 MED of UV radiation, after which chromameter reading were takento determine the degree of erythema and biopsies were taken to determinethe activity of type I collagenase and the 92 kDa gelatinase. Theresults shown in FIG. 9 show that both melatonin and vitamin Esignificantly reduced the erythema when compared with vehicle-treatedUV-exposed skin. Accordingly, while melatonin and vitamin E may beconsidered antioxidants, we have found that they provide a goodanti-sunburn sunscreen effect. Also, as shown by the results shown inFIG. 10, melatonin and vitamin E did not function to inhibit theincreased MMP activity in UV-exposed human skin.

EXAMPLE 4 Effect of UV Exposure after Topical Pretreatment II

Another theory for causes of photodamage relates to the generation ofreactive oxygen species (ROS) and other free radicals by UV radiation,because UV radiation is known to create free radicals. Accordingly, weinvestigated whether such “antioxidants” as vitamin C (ascorbic acid),N-acetyl cysteine (NAC), and 2-furildioxime (FDO), as well as aspirin(acetyl salicylic acid, ASA), had any effect on erythema or photodamagevia MMP induction.

Volunteers were pretreated 16 hours prior to exposure, the exposure andbiopsies being performed as described in the previous examples. In oneexperiment, the volunteers' skin was pretreated with vehicle alone orwith 5% ASA or with 3.5% vit. C. and tested using a chromameter forerythema and by zymography for collagenase activity. After a one (1) MEDexposure, FIG. 9 shows that pretreatment with aspirin or vitamin Creduced the UV-induced erythema upon a one (1) MED exposure from that ofuntreated (vehicle-only-treated) skin, with aspirin providing about a30% reduction in erythema versus that achieved by vitamin C, about 30%less than untreated skin. However, when the biopsies were evaluated forcollagenase activity, the results of which are shown in FIG. 10, theaspirin-treated skin evidenced a greater collagenase activity thanuntreated skin, and vitamin C provided about a 25% reduction incollagenase activity with respect to untreated skin. Again, thesesurprising results show that erythema is not correlatable toMMP-mediated UV-induced photodamage to human skin. In fact, looking onlyat erythema, one may be tempted to use aspirin, but these results showthat aspirin has no protective effect on photodegradation of skin asmediated by type I collagenase.

This same general experimental protocol was repeated at an exposure oftwo (2) MED using the vehicle alone, or with 20% NAC, or with 5% FDO,which compounds were also applied to the volunteers much prior toexposure. FIG. 9 depicts the results of the erythema analysis for thesecompounds, and shows that FDO completely inhibited erythema, while NAChad no effect (i.e., the same as the vehicle-treated skin). Unexpectedlyagain, however, analysis of type I collagenase activity at these sameexposure levels, as shown in FIG. 10, evidences that NAC providedsignificant protection against collagenase activity, while FDO providedsome protection against MMP induction.

EXAMPLE 5 Pretreatment Time Dependency

In addition to the general unpredictability of determining whether agiven compound will inhibit erythema and/or MMP-mediated degradation ofthe skin after exposure to UV radiation, we have also discovered thatthere can be a time-dependent effect of the protection.

Volunteers' skin was exposed to one (1) MED using the four bulb set andwas pretreated with vehicle alone, or with 20% NAC, either four hours orseven hours prior to exposure. Following exposure, chromameter andzymography analyses were performed as previously described.

NAC provided no anti-redness effect on UV-exposed skin, regardless ofthe duration of pretreatment. FIGS. 11A and 11B show, in comparison withthe results shown in FIG. 9, the unexpected effect on type I collagenaseafter pretreatment with NAC and exposure to 1 MED. A seven hourpretreatment with NAC provided an inhibitory effect on the UV-inducedincrease in the 92 kDa gelatinase (FIG. 11A) and collagenase (FIG. 11B)activities when compared with untreated skin (which showed over 150%increase in collagenase activity), whereas a four hour pretreatment wasineffective.

EXAMPLE 6 Effect of Commercial Sunscreens

We also evaluated commercially available sunscreens for their effect onUV-induced erythema and collagenase activity. Volunteers' skin waspretreated with the standard vehicle and with three sunscreens (ondifferent areas of skin): an SPF (sun protection factor) 15 compositionincluding ethylhexyl p-methoxycinnamate and oxybenzone; an SPF 30composition stated on the packaging to provide UVA and UVB protectionand comprising octocrylene (10%), octyl methoxycinnamate (7.5%), andoxybenzone (6%); and an SPF 50 composition stated on the packaged toprovide UVA and UVB protection and comprising higher amounts of the samecomponents as the SPF 30 composition.

After pretreatment with the vehicle and the sunscreen on different areasof skin, and then exposure to two (2) MED, the volunteers' skin wasevaluated for erythema. As the results in FIG. 7 show, all of thecommercially available sunscreens provided excellent protection againstUV-induced erythema; there was essentially no redness in comparison withunexposed skin.

EXAMPLE 7 Physiological Effect of Regular Suberythemal UV Exposure

We examined the effect of repeated suberythemal UV dosing on theinduction of MMPs, specifically type I collagenase and the 92 kDagelatinase, in vivo. Volunteers were irradiated at 0.5 MED on fourseparate sites, with each cite receiving one, two, three, or four UVexposures, the exposures being separated by 48 hour intervals. Skin wasbiopsied from each volunteer twenty four hours after the last exposure,including skin from a non-irradiated area (used as control), andanalyzed for MMP activity. As shown in FIG. 3, collagenase andgelatinase activities were elevated 2.2-fold and 4.4-fold, respectively,after a single UV exposure, and remained elevated at essentially thesesame levels upon repeated exposure every other day for four days.

While we have shown that application of a retinoid (especiallytrans-retinoic acid, tRA) can, post-UV exposure, decrease MMP activityin the skin, we also investigated the effect of pretreatment with tRAbefore exposure. Treatment of skin with tRA did not alter the low basallevels of collagenase, the 92 kDa gelatinase, or stromelysin, andsubsequent irradiation with UV lead to substantial reduction in thelevel of these three MMPs in retinoid pretreated skin in comparison withunpretreated skin. Volunteers were exposed to various UV doses rangingfrom 0.01 to 2 MED and biopsies taken from these and an unexposed area.As shown in FIGS. 2, 3, and 14A-14D, stromelysin-1 was induced withineight hours after exposure at a quite low, suberythemal, exposure level;induction was clearly evident at 0.1 MED. After these exposures, 0.1%tRA was applied daily for three days to the exposed areas and biopsieswere taken again. As shown in FIG. 13B, tRA did cause a significantreduction in stromelysin-1 protein.

EXAMPLE 8 Effect of UV Wavelength on MMP Induction

We investigated the effect of pretreatment of skin with a known UVAblocker on both erythema and MMP activity after exposure to 2 MEDs of UVradiation. In particular, we used PARSOL® 1789 (also known as PARSOL A)brand of 4-t-butyl-4′-methoxydibenzoylmethane, which is described inU.S. Pat. No. 4,387,089 (the disclosure of which is incorporated hereinby reference). (PARSOL MCX and PARSOL MOX are trademarks for2-ethylhexyl p-methoxycinnamate, a UVB blocker commonly used incommercial sunscreen, and disclosed in U.S. Pat. No. 4,713,473, thedisclosure of which is incorporated herein by reference). The absorbancecharacteristics of PARSOL® 1789 over the UVA₁, UVA₂, and UVB wavelengthsis shown as the dotted line in FIG. 8A. As shown therein, this compoundis especially useful at blocking UVA₂ radiation and somewhat effectiveat blocking UVA₁, radiation. The shaded line shows the wavelengths ofnatural erythemogenic UV radiation; as seen, erythema is causedprimarily by UVB radiation.

In a set of experiments, volunteers had areas of unexposed skinpretreated with a vehicle alone or with 5% of the PARSOL® 1789 UVAblocker. These pretreated areas were exposed to about 2 MED of UVradiation, and later tested for erythema, and biopsied to test foractivity of the 92 kDa gelatinase and the presence of cJUN protein.

FIG. 8B shows the results of post-exposure testing for sunburn, in whichPARSOL® 1789 pretreated skin was not protected from sunburn induced byUV exposure. Based on the significant blocking of UVA radiation byPARSOL® 1789, these results confirm that UVB radiation is the primaryculprit in sunburn.

FIG. 12 shows the results of in vivo activity assays of the gelatinasein the volunteers' skin, which activity was significantly reduced inUV-exposed PARSOL® 1789-pretreated skin when compared with UV-exposedvehicle-treated skin. In fact, the gelatinase activity in the UVAblocker-treated skin was not significantly different fromvehicle-treated unexposed skin. These results show that UVA is a clearcause of UV-mediated MMP induction in UV-exposed skin. Accordingly, onlycertain wavelengths of UV radiation are prone to causing photoaging andphotodegradation of skin. Thus, our invention includes the prevention ofphotoaging by the use of a UVA-blocking sunscreen.

EXAMPLE 9

As shown above, there can be a delay between the application of theactive ingredient to the skin and its ability to inhibit MMPs or itsprecursors in vivo. Shown in FIG. 15A are the results of time coursestudy of the topical application of RA and its effects on the inhibitionof collagenase, gelatinase, and cJUN protein upon exposure to UVradiation.

Volunteers were pretreated with vehicle alone, or with vehicle plus 0.1%RA at 7, 16, 24, and 48 hours prior to exposure of the skin to 2 MED ofUV radiation, and 24 hours after exposure biopsies were taken from theexposed portion of the volunteers' skin. As shown in FIG. 18A,pretreatment with the vehicle alone 24 hours prior to exposure providesa baseline activity for the collagenase and gelatinase. Pretreatmentwith RA seven hours prior to exposure did not yield activities for thecollagenase or gelatinase significantly different than the vehiclealone. At 16 hours pretreatment, the collagenase activity is not muchdifferent from that with pretreatment with the vehicle alone, but thegelatinase activity is clearly decreased. At 24 hours pretreatment, boththe collagenase and gelatinase activities are significantly lower thantheir activities when only the vehicle was used. At 48 hourspretreatment, the collagenase and gelatinase activities are reduced evenfurther.

Investigation was also made to determine whether the amount of cJUNprotein in skin exposed to UV radiation changed depending on whether (1)the skin was pretreated 48 hours before exposure with (a) the vehiclealone or (b) with RA in the vehicle, and (2) at 8 hours before exposurewith RA dispersed in the vehicle. As seen from FIG. 15B, pretreatmentwith RA eight hours before exposure did not cause any change in theamount of cJUN in the skin compared with pretreatment (48 hrspre-exposure) with the vehicle alone. On the other hand, pretreatmentwith RA 48 hours before exposure yielded a significant reduction in theamount of CJUN protein in the skin. In view of the pathway shown in FIG.1, an increase in the amount of cJUN in the skin would be expected toresult in increased AP-1 concentrations and, inevitably, an increase inMMPs with concomitant tissue degradation.

In view of these results, when a retinoid is used as the activeingredient to inhibit photoaging, it is preferred to apply the retinoidto skin more than 8 hours, more preferably at least 16 hours, even morepreferably at least 24 hours, and even up to 48 hours prior to exposureto UV radiation. As shown in our prior application 588,771, theactivities of collagenase and gelatinase can take a significant amountof time to increase from their base levels, up to 48 hours, afterexposure to UV radiation. The results shown in this example now indicatethat it can also take a not insignificant amount of time for topicallyapplied retinoids to down regulate the MMP pathway. Thus, a preferredmethod for inhibiting photoaging is using the present compositions theday prior to the day during which protection is desired, and mostpreferably the present compositions are used daily, so that photoagingis always inhibited (especially when, as we have shown, that incidental,suberythemal UV doses up-regulate MMP activity).

METHODS USED IN THE EXAMPLES

The references noted in this section are incorporated herein byreference.

Preparation of skin supernatants for biochemical analysis. Skin sampleswere ground by mortar and pestle under liquid nitrogen, and homogenizedin a Dounce tissue grinder in buffer containing 10 mM Hepes, 1 mM EDTA,5 mM EGTA, 10 mM MgCl₂, 50 mM glycerophosphate, 5 mM NaVO₄, 2 mM DTT,0.5 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 10 μg/mlpepstatin, and 0.5% NP-40. Homogenates were centrifuged at 14,000 g for15 min., and supernatants were collected and used for biochemicaldeterminations as described herein.

Matrix metalloproteinase assays. Tissue pieces were frozen in liquidnitrogen immediately after biopsy, homogenized in 20 mM Tris HCl (pH7.6) plus 5 mM CaCl₂, and centrifuged at 3000× g for 10 minutes toremove particulates. Ability to release soluble radioactive fragmentsfrom 3 H-labeled fibrillar Type I collagen (described by Fisher, G. J.,et al., Nature, 379, 335-339 (1996) and Hu, C-L, et al., Analytic.Biochem, 88, 638-643 (1978)) was used as a measure of collagenolyticactivity. Tissue extracts were incubated for 3 hours with 1 mMaminophenyl mercuric acetate (APMA) to convert the inactive form of thematrix metalloproteinase into an active form. Subsequently, 0.2 μCi ofcollagen substrate (NEN-DuPont, Boston, Mass.) was incubated for 24hours with 50 μl of tissue extract. At the end of the 24-hour incubationperiod, the samples were centrifuged at 12,000×g for 10 minutes topellet the intact protein. Radioactivity remaining in the supernatantfluid was then measured and from this, the percentage of substratehydrolzyed was determined.

Gelatin zymography (Varani et al., op. cit.) was used to assess MMP-2(72-kD gelatinase; gelatinase A) and MMP9 (92-kD gelatinase; gelatinaseB) activity. Tissue extracts were electrophoresed in an 8.5%SDS-polyacrylamide gel containing 1 mg/ml of gelatin. Afterelectrophoresis, the SDS was removed by three sequential washes in 1%Triton X-100. The first two washes were for 20 minutes each and the lastwas overnight. Quantitation of hydrolysis zone width was done by laserdensitometry.

c-Jun kinase activity assay. c-Jun activity in skin supernatants wasdetermined by solid phase kinase assays (as described, e.g., by M. Hibiet al., “Identification of an oncoprotein and UV-responsive proteinkinase that binds and potentiates the c-Jun activation domain,” GenesDev., 7:2135-2148 (1993)).

Northern analysis of RNA. Total RNA (e.g., for c-Jun) was isolated fromskin samples by guanidinium hydrochloride lysis and ultracentrifugation(as described by G. J. Fisher et al., “Cellular, immunologic andbiochemical characterization of topical retinoic acid-treated humanskin,” J. Investig. Dermatol., 96:699-707 (1991)). Northern analysis oftotal RNA (40 μg/lane) with randomly primed ³²P labelled cDNA probes forthe particular mRNA to be determined were performed as described by G.J. Fisher et al. (in “All trans retinoic acid induces cellularretinol-binding protein in human skin in vivo,” J. Investig. Dermatol.,105:80-86 (1995)).

Western analysis of proteins. Jun proteins were detected in nuclearextracts from human skin by Western analysis as described by G. J.Fisher et al. (in “Immunological identification and functionalquantitation of retinoic acid and retinoid X receptor proteins in humanskin,” J. Biol. Chem., 269:20629-20635 (1994)). Immunoreactive proteinswere visualized by enhanced chemiluminescence detection and quantifiedby laser densitometry, or by enhanced chemifluorescence detection andquantified by a Storm imager (Molecular Dynamics, Palo Alto, Calif.).

Chromameter: erythema (skin reddening) was determined 24 h post-exposureusing a commercially available Minolta chromameter (chromameter CR200,model 94401085).

The foregoing description is meant to be illustrative and not limiting.Various changes, modifications, and additions may become apparent to theskilled artisan upon a perusal of this specification, and such are meantto be within the scope and spirit of the invention as defined by theclaims.

What is claimed is:
 1. A composition for inhibiting photoaging of humanskin, comprising a combination of a UVA1 blocker preventing penetrationof UV light having a wavelength between about 360 nm and 400 nm, a UVBblocker preventing penetration of UV light having a wavelength ofbetween about 300 and 320 nm, an amount of an MMP inhibitor effective toinhibit the UV-induction of increased MMP activity, and a topicallyacceptable carrier.
 2. The composition of claim 1, wherein the MMPinhibitor is a retinoid.
 3. The composition of claim 1, furthercomprising a compound selected from the group consisting of N-acetylcysteine, FDO, vitamin C, and mixtures thereof.
 4. The composition ofclaim 1, wherein the UVA1 blocker is4-t-butyl-4′-methoxydibenzoylmethane or a derivative thereof.
 5. Thecomposition of claim 1, wherein the UVB blocker is an oxybenzone or amethoxycinnamate.
 6. The composition of claim 1, formulated as a lotion,cream, ointment, water-based liquid, oil-based liquid, or sprayableliquid.
 7. The composition of claim 2, wherein the retinoid is retinoicacid or retinol.
 8. The composition of claims 1, wherein saidcomposition further comprises at least one additional compound selectedfrom the group consisting of tetracycline and derivatives thereof,dermatologically acceptable triazoles and derivatives thereof, acompound that inhibits the cytochrome P-450 mediated-metabolism ofretinoic acid, and compatible mixtures thereof.
 9. The composition ofclaim 1, provided as a day and night combination of compositions forinhibiting photoaging of human skin induced by UV radiation, comprising:a first composition for use during the day and comprising thecomposition defined by claim 1; and further comprising a secondcomposition for use during the evening, said second compositioncomprising an MMP inhibitor in a dermatologically acceptable carrier;wherein said first and second compositions are provided together andpackaged separately from each other, and further comprising instructionsfor the use of said first and second compositions.
 10. The day and nightcombination of claim 9, wherein the MMP inhibitor in each of said firstand second compositions is selected independently from the groupconsisting of a retinoid, N-acetyl cysteine, 2-furildioxime, vitamin C,and mixtures thereof, in a dermatologically acceptable carrier.
 11. Thecomposition of claim 10, where the retinoid in the first composition,the second composition, or both, is selected independently from thegroup consisting of retinoic acid, retinol and mixtures thereof.